Microscopy Boot Camp 2009 2009/08/25 Nikitchenko Maxim Baktash Babadi.

Microscopy

Boot Camp 20092009/08/25

Nikitchenko Maxim

Baktash Babadi

Plan of the lecture

• Basic properties of light• Light/matter interaction• Wide-field microscopy• Scanning microscopy• EM• Ultra-high resolution microscopy• Dyes

Part 1

Part 2

Part 3

Corpuscular/wave dualism

www.olympusmicro.com

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Diffraction

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Basic electromagnetic wave properties

• Amplitude• Wavelength• Frequency• Phase• Polarization

hyperphysics.phy-astr.gsu.edu

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

)sin(0 tkzEE

Polarization

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Light/matter interaction

• Particles point of view: Absorption

Emission Scattering

• Waves point of view:Refraction

ReflectionAbsorptionDiffraction (Change of Phase and Polarization)

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Optics of a thin lens (1)

Focus

d F

F CC F

C=2F

0F

dThin Lens:

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Optics of a thin lens (2)• Three different scenarios:

F 2F2F F

F 2F2F F

F 2F2F F

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Optics of a thin lens (3)

f

p q

F CC F

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Compound Microscope

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Basic optical structure of a microscope

objective

specimen

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

eyepiece

Specimen Illumination System

• Parts– Specimen plane

– Condenser

– Diaphragm

– Light Source

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Microscope Illumination Conditions:

• Critical illumination– The condenser focuses

the light onto the specimen plane

Filament image effect

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Köhler illumination

• The specimen is illuminated homogenously

• The specimen and the images of the light source are in different planes

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Types of Microscopy

• Bright Field (absorption)

• Dark Field (scattering)

• Phase-contrast (phase change)

• Polarization (scattering by birefringent specimen)

• Differential interference contrast (DIC) (gradients of optical thickness)

• Fluorescent (frequency change as a result of absorption/emission by fluorophores)

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Dark field Microscopy

uses the difference in scattering abilities

block out the central light rays (leave oblique only)

Result: only highly diffractive and scattering structures are seen

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Dark-Field example

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

uses the λ/4 phase change when light passes through thin structures

Similar oblique illumination to the Dark Field method

The specimen diffracts some of the light that passes through it and introduces phase lagging λ/4

A phase difference (λ/2) is introduced between background and diffracted light (using phase plate) → destructive interference

Phase Contrast Microscopy

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Phase Contrast Microscopy

• Suitable for unstained specimens

Human glial cells

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Polarization MicroscopyUses polarization property of

light and birefringence• Polarizer polarizes light• Analyzer passes only the light

with polarization perpendicular to the source light

Birefringent material introduces 2 perpendicularly polarized components, propagating at different speed in the specimen → Δφ

• Constructive interference following analyzer is possible only for phase shifted light

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Polarized microscopy example

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Differential Interference Contrast (DIC) (Nomarski optics)

• Addon to the polarization microscopy

• Wollaston prism generates 2 || beams, π/4 polarized to polarizer and laterally displaced (this is the difference to polarization microsc., endowing optical density gradient sensitivity)

• The rest is similar to pol. Micr. (except for 2nd Wollaston prism)

• Result: good for edge detection

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Nomarski optics principle

polarizer

Beam

Splitter

shear

condenser

specimen

objective

combiner

analyzer

intensity

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

DIC example

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Fluorescent Microscopy (1)

• Fluorescence– Emission light has longer wavelengths than

the excitation light: Stokes shift.

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Fluorescent Microscopy (2)

• Types of Fluorescence– Auto-Fluorescence (Plants, Fungi,

Semiconductors, etc)– Fluorescent dyes

• Fluorochromes (Flurescein, Acredine Orange, Eosin, Chlorophyll A, … )

• Genetically coded (GFP, YFP,…)

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Fluorescent Microscopy (3)

• The basic task of the fluorescence microscope:– Illuminate the specimen with excitation light– Separate the much weaker emission light

from the brighter excitation light. – Only allow the emission light to reach the

eye or other detector.– The background is dark, the fluorescent

objects are bright

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Epi-Fluorescent Illumination

• The emission light does not pass through the specimen

• The objective acts as the condenser

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Fluorescent Microscopic images (1)

Human cortical neurons Human brain glioma cells

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Fluorescent Microscopic images

Fluorescence/DIC combination, cat brain tissue infected with Cryptococcus

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Brainbow

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Selection of the microscopeIs it thin? (<50 microns)

Is it reflective?e.g. gold, silver

Is it fluorescent?

Confocal

Epi-fluorecenceIs it fluorescent?

Is it colored, densely contrasted or stained?

Is it transparent?

Bright field

Phase contrast, DIC

Is it reflective?e.g. gold, Silver

Is it birefringent? Polarization

Dark field

no

no

no

no

no

noyes

yes

yes

yes

yes

yes

yes

Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection

Rubbi, C.P., 1994